The long-term goal of my research is to define the roles of the nonstructural 4B (NS4B) protein in infectious HCV production and virus-induced liver cancer. Reported NS4B functions include membranous web formation, GTPase activity and RNA binding, all of which are required for HCV replication. However, we know little about NS4B interaction with itself as well as viral/host partners and how these interactions affect HCV replication. Thus, the objective of this application is to get a better insight into the contribution of NS4B to HCV production. NS4B is an integral membrane protein whose transmembrane domains (TMDs) are thought to merely insert the protein into host membranes. However, our recent results suggest that NS4B TMDs have a more dynamic role. Additionally, our lab and others have found that the highly conserved NS4B C-terminal domain (CTD) is required for HCV replication and plays a crucial role in the organization of the replicase-enclosing membranous web. However, how the CTD facilitates membranous web formation remains a mystery. Based on our current findings, we propose that: i) The TMD helices are engaged in intra/intermolecular interactions, thus contributing to NS4B structure and function, ii) The CTD dimerizes and acts in part as a SNARE protein complex to facilitate membrane fusion and hence web formation. Through a better understanding of how NS4B facilitates HCV replication, new targets for drug development could be uncovered. We will pursue the following aims: Aim 1. Define the molecular mechanism whereby NS4B TMD helices promote HCV genome replication. As a scaffolding protein, NS4B requires protein interactions to remain anchored into a specific site in the membrane. In this aim, we will: [1] use mutagenesis to define the role of the TMD helices in NS4B interactions, activity, subcellular distribution and virus production;[2]: disrupt TMD helix interactions and examine their effect on NS4B properties and JFH1 production;[3] use suppressor screens to identify intra- and intermolecular interactions involving NS4B TMDs. Aim 2. Investigate how NS4B organizes functional HCV replication complex. Once NS4B-bound vesicles leave the ER membrane, they coalesce. Thus, we propose that NS4B acts in part as a SNARE protein complex to facilitate membrane fusion leading to web formation. To test this hypothesis, we will: [1] Examine the role of NS4B CTD-CTD interaction in NS4B web formation and examine how expression of the CTD impacts JFH1 production;[2] Study the impact of the CTD mutations on NS4B RNA binding and GTPase activity;[3] Perform suppressor screens with the CTD mutant viruses to identify intra/intermolecular NS4B interactions;[4] Identify NS4B host partners. Aim 3. Decode the significance of NS4B interaction with NS3 protein. Does NS4B-NS3 interaction regulate NS4B GTPase or NS3 protease/helicase activity? In this aim, we will engineer constructs with various NS3 and NS4B domains to delineate their roles in NS4B-NS3 interaction, [1] in vivo and, [2] in vitro. We will also [3] use purified proteins to determine whether NS3 and NS4B have any regulatory effect on each other[unreadable]s activity.